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Biomedical subjects

G Feriotto

Publications and source records attributed to G Feriotto.

At least 37 records · Page 2Linked to original sources
PubMed1996

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin. Disruption of nuclear factor.DNA complexes and inhibition of in vitro transcription.

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

Base Sequence↗
PubMed1995

Binding of distamycin and chromomycin to human immunodeficiency type 1 virus DNA: a non-radioactive automated footprinting study.

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we studied sequence-selectivity of the binding of chromomycin and distamycin to DNA by performing DNase I footprinting and analysis of the cleaved fragments by the Pharmacia ALF DNA Sequencing System. As a model system we employed the long terminal repeat of the human immunodeficiency type 1 virus. The main conclusion of our experiments is that automated analysis of DNase I footprinting is a fast and reliable technique to study drugs-DNA interactions. The results obtained suggest that distamycin and chromomycin differentially interact with the long terminal repeat of the human immunodeficiency type 1 virus; this differential binding depends upon the DNA sequences recognized. The data presented are consistent with a preferential binding of distamycin to DNA sequences of the binding sites of nuclear factor kappa B and transcription factor IID. By contrast, distamycin exhibits only weak binding to DNA sequences recognized by the promoter-specific transcription factor Sp1. Unlike distamycin, chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1.

Antibiotics, Antineoplastic↗
PubMed1995

Differential effects of distamycin analogues on amplification of human gene sequences by polymerase-chain reaction.

In this report we analyse the effects of distamycin and five distamycin analogues on amplification by polymerase-chain reaction (PCR) of two gene sequences displaying a different A+T/G+C content. The first was a 5' region of the human oestrogen receptor (ER) gene, containing a (TA)26 stretch; the second was a CG-rich sequence of the human Ha-ras oncogene. The results obtained unequivocally demonstrate that the addition of one pyrrole ring significantly improves the ability of distamycin derivatives to interfere with PCR-mediated amplification of the human ER genomic region carrying a (TA)26 stretch. The distamycin analogues analysed differ in the number of pyrrole rings and in the presence of an N-formyl, an N-formimidoyl or a retroamide group at position X1. Among compounds carrying the same number of pyrrole rings, those carrying an N-formyl or an N-formimidoyl group retain a similar inhibitory activity. The retroamide analogues, on the contrary, are much less efficient in inhibiting PCR-mediated amplification of the 5'ER region. With respect to sequence selectivity both distamycin and distamycin analogues exhibit a sequence preference, since they do not inhibit PCR amplification of Ha-ras CG-rich gene regions, with the exception of a distamycin analogue carrying four pyrrole rings.

Base Composition↗
PubMed1995

Sequencing of an upstream region of the human HLA-DRA gene containing X' and Y' boxes.

In this paper we report the characterization of a newly sequenced 5' upstream region of the human HLA-DRA gene. We performed (i) search for transcription factor motifs, (ii) analysis of CpG display and observed/expected frequency ratios, (iii) search for regions homologous to the 5' upstream sequences of the murine EA gene, (iv) DNase I footprinting experiments and (v) electrophoretic mobility shift assays. Our results demonstrate the existence, in the HLA-DRA gene, of Y' and X' boxes highly homologous to the Y and X boxes present in MHC class II genes, but oriented in the opposite direction. These Y' and X' boxes have been conserved during the molecular evolution of both human HLA-DRA and murine EA genes. DNase I footprinting and gel retardation experiments suggest that the X' and Y' boxes of the HLA-DRA upstream gene region are specifically recognized by nuclear proteins that also bind to the X and Y boxes of the HLA-DRA proximal promoter, respectively.

Animals↗
PubMed1995

Chromatography in DNA radiolabeling: hands-off automation using a robotic workstation.

The experiments described in the present paper were performed in order to determine whether the Biomek-1000 (Beckman Instruments, Fullerton, CA, USA) automated laboratory workstation can be used in a fully automated DNA labeling method followed by automated gravity-driven size exclusion purification of molecular probes. To this aim, we performed random oligodeoxyribonucleotide priming of a HIV-1 LTR probe that was used for molecular hybridization to Southern blotted polymerase chain reaction products. The results obtained demonstrate that the automatically labeled probe can be efficiently purified by automated and gravity-driven Sephadex G-50 chromatography, without any major changes in hybridization property. This robotic methodology can be used in several procedures employing radioisotope labeling.

Base Sequence↗
PubMed1995

Alteration of the expression of human estrogen receptor gene by distamycin.

The effects of distamycin on the expression of the estrogen receptor gene were determined in the MCF7 human breast cancer cell line. Estrogen receptor (ER) RNA transcripts were analyzed by Northern blotting and RT-PCR using specific oligonucleotides for the 5' upstream region and for ER cDNA. After ex vivo distamycin treatment of the cells the expression of the canonical ER mRNA isoform of 6.3 kb is strongly inhibited, without appreciable alteration of the accumulation of 5' upstream ER mRNA isoforms. These results suggest that distamycin alters the transcriptional activity of the ER gene causing a change in the ratio between the canonical transcript and other isoforms containing 5' upstream regions.

Antiviral Agents↗
PubMed1994

Distamycin analogues with improved sequence-specific DNA binding activities.

In the present study we have investigated the effect of unprecedented chemical modifications introduced in the distamycin molecule, with the aim of assessing their ability to interfere with sequence-specific DNA-protein interactions in vitro. By using an electrophoretic mobility shift assay, we have been able to identify novel distamycin analogues with improved displacing abilities on the binding of octamer nuclear factors to their target DNA sequence. While variations in the number of pyrrole rings and/or reversion of an internal amide bond result in distamycin-like compounds with identical or very similar properties, the reversion of the formamido into a carboxyamido group or its replacement with the charged formimidoyl moiety significantly improves the ability of the resulting novel distamycin derivatives to compete with OCT-1 (octamer 1 nuclear factor) for its target DNA sequence. Tissue-specific octamer-dependent in vitro transcription is similarly affected by these chemical modifications, suggesting that the ability of distamycins to bind octamer sequences has a direct influence on the functional state of octamer-containing promoters. These data represent an initial, successful attempt to rationalize the design of DNA binding drugs, using distamycins as a model.

Amides↗
PubMed1994

Binding of Epstein-Barr virus nuclear antigen 1 to DNA: inhibition by distamycin and two novel distamycin analogues.

Modulation of the interaction between cellular or viral transcription factors and target DNA sequences may represent a potential experimental strategy to control proliferation of neoplastic cells as well as virus DNA replication. Distamycin represents a likely candidate to mediate such modulation by pharmacological means. In order to obtain more detailed information on structure-activity relationships of these compounds, we have analysed the effects of distamycin and two distamycin analogues on the binding of a recombinant protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1) to its target sequence of Epstein-Barr virus, containing the 12 bp palindromic consensus TAGCATATGCTA. The sequence selectivity in the binding of distamycin to DNA was evaluated by footprinting experiments, while the effects of distamycins on DNA-protein interactions was analysed by means of electrophoretic mobility shift assay. The data presented in this paper suggest that distamycin and its analogues differentially inhibit the interaction between DNA-binding proteins and target DNA sequences.

Antigens, Viral↗
PubMed1994

Sequence-specific recognition of the HIV-1 long terminal repeat by distamycin: a DNAase I footprinting study.

Pharmacological modulation of the interaction between transcription factors and target DNA sequences of cellular and viral genes could have important effects in the experimental therapy of a large variety of human pathologies. For instance, alteration of the DNA/protein interaction might be among the molecular mechanisms of action of DNA-binding drugs, leading to an inhibition of the expression of genes involved in the control of in vitro and in vivo growth of neoplastic cells and virus DNA replication. Natural oligopeptides, such as distamycin, are powerful inhibitors of the interaction between nuclear factors and target DNA sequences and, therefore, have been proposed as compounds retaining antibiotic, antineoplastic and antiviral properties. In this study we performed DNAase I footprinting analysis using a PCR product mimicking a region of the long terminal repeat (LTR) of the human immunodeficiency type 1 (HIV-1) retrovirus. The data obtained suggest that distamycin binds to different regions of the HIV-1 LTR depending on the DNA sequence. Electrophoretic mobility shift assays using both crude nuclear extracts from the Jurkat T-lymphoid cell line and the recombinant proteins transcription factor IID and Sp1 suggest that distamycin differentially inhibits the interaction of these two proteins with their specific DNA target sequences, in good agreement with the results obtained by DNAase I footprinting analysis.

Base Sequence↗
PubMed1994

A chromatographic procedure for fully automated isolation of DNA from human whole blood.

Isolation of DNA from peripheral blood constitutes a fundamental step in the molecular diagnosis of genetic disorders, analysis of mutations of oncogenes and detection of nucleic-acid sequences of pathogenic organisms in clinical samples. We investigated whether dextran sedimentation of blood followed by the chromatographic isolation of DNA could be applied to the Biomek-1000 automated laboratory workstation in order to develop a fully automated method of DNA isolation from whole blood. Under our experimental conditions, the Biomek-1000 could perform fully automated DNA isolation from seven human whole blood samples in less than 1.5 h. Our data indicate that the DNA isolated from human blood in this way is suitable for polymerase chain reaction.

Autoanalysis↗
PubMed1994

Polymerase-chain reaction as a tool for investigations on sequence-selectivity of DNA-drugs interactions.

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.

Animals↗
PubMed1994

Capillary electrophoresis: detection of hybridization between synthetic oligonucleotides and HIV-1 genomic DNA amplified by polymerase-chain reaction.

The polymerase chain reaction (PCR) is one of the most efficient techniques for measuring the viral load of HIV-infected samples. Determination of the specificity of PCR products is usually based on Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes specific for sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) for identification of HIV-1 and HTLV-I PCR products appears interesting in light of its reproducibility, sensitivity and because it is fast and suitable for detection of DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of a HIV-1 oligonucleotide probe to single-stranded DNA obtained by unbalanced PCR is detectable by capillary electrophoresis. This enabled us the application of a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. This procedure is simple, reproducible and is suggested as an integral part of automated diagnostic systems based on the use of laboratory work stations for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Base Sequence↗
PubMed1994

Differential inhibition of DNA/protein interactions by aromatic amidines with 2, 3 and 4 benzamidine residues.

We have recently reported that aromatic polyamidines are powerful inhibitors of in vitro proliferation of tumour cell lines and in vivo tumorigenicity of melanoma cells xenografted into nude mice. Interestingly, we have found that tetrabenzamidines are able to bind DNA, and to inhibit the interaction between transacting factors and specific target DNA sequences. In order to obtain more detailed information on structure-activity relationships, we have analysed the effects of different aromatic polyamidines on the binding of a recombinant protein, the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), to the target sequence of EBV DNA, containing the 12 bp palindromic consensus TAGCATATGCTA. The results obtained suggest that aromatic polyamidines inhibit the interactions between DNA-binding proteins and target DNA sequences with different efficiency, depending (i) on the number of amidine residues and (ii) on the presence of halogen substitutions (Cl, Br or I) on the benzene rings of tetra-benzamidine molecules.

Amidines↗
PubMed1993

Methylation state of the human HLA-DRA gene in T-lymphocytes and B-lymphocytes of transgenic mice. Lack of methylation at one 5'-GCGC site is not required for gene expression.

A consistent lack of DNA methylation at one or both of two GCGC (Hha I) restriction sites in the 5' region of the HLA-DRA gene has been previously documented by the use of methyl-sensitive restriction enzymes in human cells and tissues, irrespectively of their expression of DR alpha products. Evidence presently available, however, does not exclude that a lack of methylation in this region, although not sufficient, might be necessary for gene expression. In this report, we show that only one of the 5'-GCGC sites is protected, although less efficiently than in man, from CG-->mCG modifications in tissues and cells of transgenic mice carrying an expressed single copy of the HLA-DRA gene/diploid genome. We demonstrate that the two 5' GCGC sites of the HLA-DRA transgene are fully methylated in DR alpha- splenocytes (more than 80% T-lymphocytes), while one of them (the most 5' site) is not methylated in a fraction of DR alpha+ splenocytes (more than 95% B-lymphocytes). These results provide evidence that absence of DNA methylation in the 5' region is not necessary for, but might be associated with and possibly secondary to the expression of the DRA gene.

Animals↗
PubMed1993

A new automated method for isolation of genomic DNA from eukaryotic cells.

In this study we determine whether the BIOMEK-1000 Laboratory Workstation (Beckman Instruments, Fullerton, CA) can be used to isolate genomic DNA from eukaryotic cells. The results obtained demonstrate that DNA isolated by BIOMEK-1000 is suitable for amplification of genomic sequences by PCR. Our data prove that automated chromatographic DNA isolation employing the BIOMEK-1000 could be used in molecular diagnosis of human pathologies.

Automation↗
PubMed1993

Use of an automated laboratory workstation for isolation of genomic DNA suitable for PCR and allele-specific hybridization.

In this paper we describe the isolation of genomic DNA by using anion exchange chromatography performed on a Biomek 1000 Automated Laboratory Workstation. This procedure allows the automated isolation of DNA suitable for most molecular analyses employed in diagnosis of genetic pathologies and infectious diseases. The genomic DNA isolated by using the Biomek 1000 was indeed found to be suitable for polymerase chain reaction and allele-specific hybridization.

Alleles↗
PubMed1992

DNA binding activity and inhibition of DNA-protein interactions. Differential effects of tetra-p-amidino-phenoxyneopentane and its 2'-bromo derivative.

In the present study are reported the differential DNA binding activity of the anti-tumor polyamidine tetra-p-amidinophenoxyneopentane (TAPP-H) and its 2'-halo derivative (TAPP-Br), and their effects on the binding of the recombinant Epstein-Barr virus (EBV) nuclear antigen to a synthetic oligonucleotide mimicking the target DNA sequence present in the EBV genome. In addition, the proliferation kinetics and cell cycle analysis of human leukemia K562 cells treated with TAPP-H and TAPP-Br are reported. The possible in vivo relationship between DNA binding affinity and cytotoxicity is also discussed.

Antigens, Viral↗